The general goal of the project is to use a single cell approach to dissect excitation-contraction (E-C) coupling pathways in the vascular smooth muscle cell. Based on progress made in this laboratory as well as in other laboratories over the last grant period, the next five years will be centered around testing the specific hypothesis that protein kinase C(PKC) can provide a "second" signalling pathway in addition to that created by calcium-dependent myosin light chain phosphorylation. The outlined studies are aimed at elucidating the details of the mechanisms involved. Alternatively, should the hypothesis not be correct, the experiments should clearly disprove it. The specific aims are: 1) to complete investigations using a newly available PKC probe to determine the distribution and agonist-induced redistribution of perinuclear PKC as well as the degree to which total PKC distribution coincides with the sum of the isoform specific antibody staining; 2) to use Western Blots in combination with immunofluorescence at a single cell level to determine the relative content and location of Ca-dependent and Ca-independent isoforms of PKC in ferret aorta and ferret portal vein. The ferret aorta is known to be able to contract in a Ca-independent manner whereas the portal vein is more Ca-dependent; 3) to test the feasibility of putative physiologic PKC substrates by using digital imaging microscopy and immunofluorescence to co-label PKC and suspected substrates (especially calponin and MAP-2 kinase) at various time points during agonist stimulation; 4) to test the hypothesis that caldesmon or calponin are involved in smooth muscle contractions in either a PKC- dependent or PKC-independent manner by testing the effect of peptide fragments of these proteins or force developed by single hyperpermeable cells; 5) to test the hypothesis that a constituitively active hydrophilic fragment of PKC (PKM) is generated in intact smooth muscle cells by comparing immunofluorescence obtained with antibodies directed at the catalytic subunit versus those directed at the regulatory subunit as well as the effect of inhibitors specific to the regulatory versus catalytic subunit; 6) to test the hypothesis that a non-crossbridge mechanism is involved in force maintenance by determining if there is a unique bell-shaped active length-tension curve in vascular smooth muscle cells. These studies will add to our understanding of the way in which normal vascular smooth muscle cells contract and eventually should aid in the understanding of the defects in pathophysiological states of altered vascular tone such as hypertension and vascular spasm.